Umio Yamaguchi

Distinct Gene Expression-Defined Classes of Gastrointestinal Stromal Tumor

PURPOSE The majority of gastrointestinal stromal tumors (GIST) can be cured by surgery alone, but relapse occurs in 20% to 40% of cases. GISTs are considered to invariably arise through gain of function KIT or PDGFA mutation of the interstitial cells of Cajal (ICC). However, the genetic basis of the malignant progression of GISTs are poorly understood. PATIENTS AND METHODS The expression levels of 54,613 probe sets in 32 surgical samples of untreated GISTs of the stomach and small intestine were analyzed with oligonucleotide microarrays. The representative GeneChip data were validated by real-time reverse transcriptase polymerase chain reaction and immunohistochemistry. RESULTS Unbiased hierarchical clustering consistently separated the 32 cases of GIST into two major classes according to tumor site. The two major classes were further separated into novel subclasses, which were significantly correlated with various pathological prognostic parameters, the frequency of metastasis (P < .05), and clinical outcome. Immunohistochemical analysis of 152 independent patients with gastric GISTs revealed that the expression of dipeptidyl peptidase IV (T-cell activation antigen CD26) protein was significantly associated with poorer overall and disease-free survival (P < .00001). CONCLUSION CD26 appears to be a reliable biomarker of malignant GISTs of the stomach. The postoperative recurrence rate of CD26-negative cases was as low as 2.0% (two of 102). Therefore, postoperative follow-up of such patients might be made less intensive. CD26 may play an important role in the malignant progression of gastric GISTs and serve as a therapeutic target.

Glut-1 expression and enhanced glucose metabolism are associated with tumour grade in bone and soft tissue sarcomas: a prospective evaluation by [18F]fluorodeoxyglucose positron emission tomography

PurposeThis study was conducted to investigate whether 18F-fluorodeoxyglucose (FDG) uptake, quantified by positron emission tomography (PET), correlates with histological variables including tumour grade, cell proliferation, cell cycle control integrity and glucose metabolism in patients with bone and soft tissue sarcomas.MethodsEighty-two patients clinically suspected of having a bone or soft tissue sarcoma underwent FDG PET within 1 week prior to operation and 63 patients (mean age 48 years, range 18–74 years) were enrolled in the complete analysis. We excluded 17 patients with pathologically confirmed benign tumours and two patients with uncontrolled diabetes or concomitant malignancy from data analysis. Maximum and average standardised uptake values (SUVs) of the primary lesion were compared with histological variables including tumour differentiation, the presence of necrosis, MIB-1 score, mitotic score, p53 overexpression, MIB-1 grade, mitotic grade and GLUT-1 expression.ResultsSignificant correlations were found between maximal and mean SUVs and MIB-1 grade, mitotic grade, MIB-1 score, tumour differentiation and mitotic score. The mean and maximal SUVs were significantly higher in tumours with p53 overexpression than in those without p53 overexpression (p<0.0001). GLUT-1-positive tumours had significantly higher mean (6.5±4.2 vs 1.1±0.2, p=0.006) and maximal SUVs (8.8±5.4 vs 1.7±0.5, p=0.005) than the GLUT-1-negative tumours. GLUT-1 intensity correlated positively with both mean (r=0.500, p<0.0001) and maximal SUVs (r=0.509, p<0.0001). Mu ltiple linear regression analysis showed a significant correlation between maximal SUV and MIB-1 grade (p<0.0001).ConclusionThe enhanced glucose metabolism, as determined by SUV, is a strong index of tumour grade in bone and soft tissue sarcomas.

Expression and Gene Amplification of Actinin-4 in Invasive Ductal Carcinoma of the Pancreas

Purpose: An invasive growth pattern is one of the hallmarks of pancreatic ductal carcinoma. Actinin-4 is an actin-binding protein associated with enhanced cell motility, invasive growth, and lymph node metastasis. Actinin-4 might play an important role in the development and progression of pancreatic cancer. Experimental Design: The expression of actinin-4 was examined immunohistochemically in 173 cases of invasive pancreatic ductal carcinoma. The copy number of the actinin-4 (ACTN4) gene was calculated by fluorescence in situ hybridization. The expression of actinin-4 was stably knocked down by short hairpin RNA, and tumorigenicity was evaluated by orthotopic implantation into mice with severe combined immunodeficiency. Results: The expression level of actinin-4 was increased in 109 (63.0%) of 173 cases of pancreatic cancer. Kaplan-Meier survival curves revealed that patients with increased expression of actinin-4 had a significantly poorer outcome (P = 0.00001, log-rank test). Multivariate analysis by the Cox proportional hazard model showed that high expression of actinin-4 was the most significant independent negative predictor of survival (hazard ratio, 2.33; P = 0.000009). Amplification (defined as more than four copies per interphase nucleus) of the ACTN4 gene was detected in 11 (37.9%) of 29 cases showing increased expression of actinin-4. Knockdown of actinin-4 expression inhibited the destructive growth of cancer cells in the pancreatic parenchyma. Conclusion: Recurrent amplification of chromosome 19q13.1-2 has been reported in pancreatic cancer, but the exact target gene has not been identified. Actinin-4 contributes to the invasive growth of pancreatic ductal carcinoma, and ACTN4 is one of the candidate oncogenes in this chromosome locus.

Differential diagnosis of gastrointestinal stromal tumor and other spindle cell tumors in the gastrointestinal tract based on immunohistochemical analysis

To confirm the usefulness of an immunohistochemical panel of antibodies for KIT (c-kit/CD117), CD34, desmin, smooth-muscle actin (SMA), h-caldesmon (HCD), S-100 protein, neuron-specific enolase (NSE), and beta-catenin, 297 mesenchymal and peripheral nerve-sheath tumors of the gastrointestinal tract and intra-abdominal locations including 211 gastrointestinal stromal tumors (GISTs), 12 leiomyomas, 18 leiomyosarcomas, 17 solitary fibrous tumors (SFTs), 14 schwannomas, and 25 desmoid-type fibromatoses (DTFs) were analyzed immunohistochemically. Consistent (100%) immunoreactivity for KIT, CD34, desmin and S-100, and nuclear accumulation of beta-catenin were detected in GISTs, SFTs, smooth-muscle tumors, schwannomas, and DTFs, respectively. Immunoreactivity for SMA, HCD, and NSE was observed in a wide range of these tumors. In addition, 418 bone and soft tissue tumors were enrolled in this study for KIT immunostaining. As a result, a limited number of these tumors were KIT positive, including synovial sarcoma that showed morphological similarity to GISTs. These findings suggest that KIT, CD34, desmin, S-100, and beta-catenin are key markers for clinical diagnosis of GISTs and other spindle cell tumors that may involve the gastrointestinal tract, whereas SMA, HCD, and NSE have only limited value.

Reduced Argininosuccinate Synthetase Is a Predictive Biomarker for the Development of Pulmonary Metastasis in Patients with Osteosarcoma

Pulmonary metastasis is the most significant prognostic determinant for osteosarcoma, but methods for its prediction and treatment have not been established. Using oligonucleotide microarrays, we compared the global gene expression of biopsy samples between seven osteosarcoma patients who developed pulmonary metastasis within 4 years after neoadjuvant chemotherapy and curative resection, and 12 patients who did not relapse. We identified argininosuccinate synthetase (ASS) as a gene differentially expressed with the highest statistical significance (Welchs t test, P = 2.2 × 10−5). Immunohistochemical analysis of an independent cohort of 62 osteosarcoma cases confirmed that reduced expression of ASS protein was significantly correlated with the development of pulmonary metastasis after surgery (log-rank test, P < 0.05). Cox regression analysis revealed that ASS was the sole significant predictive factor (P = 0.039; hazard ratio, 0.319; 95% confidence interval, 0.108-0.945). ASS is one of the enzymes required for the production of a nonessential amino acid, arginine. We showed that osteosarcoma cells lacking ASS expression were auxotrophic for arginine and underwent G0-G1 arrest in arginine-free medium, suggesting that an arginine deprivation therapy could be effective in patients with osteosarcoma. Recently, phase I and II clinical trials in patients with melanoma and hepatocellular carcinoma have shown the safety and efficacy of plasma arginine depletion by stabilized arginine deiminase. Our data indicate that in patients with osteosarcoma, reduced expression of ASS is not only a novel predictive biomarker for the development of metastasis, but also a potential target for pharmacologic intervention. Mol Cancer Ther; 9(3); 535–44

A practical approach to the clinical diagnosis of Ewing’s sarcoma/primitive neuroectodermal tumour and other small round cell tumours sharing EWS rearrangement using new fluorescence in situ hybridisation probes for EWSR1 on formalin fixed, paraffin wax embedded tissue

Background: Over 90% of Ewing’s sarcoma/primitive neuroectodermal tumour (ES/PNET) cases have the t(11;22) chromosomal rearrangement, which is also found in other small round cell tumours, including desmoplastic small round cell tumour (DSRCT) and clear cell sarcoma (CCS). Although this rearrangement can be analysed by fluorescence in situ hybridisation (FISH) using routinely formalin fixed, paraffin wax embedded (FFPE) tissues when fresh or frozen tissues are not available, a sensitive and convenient detection method is needed for routine clinical diagnosis. Aims: To investigate the usefulness of newly developed probes for detecting EWS rearrangement resulting from chromosomal translocations using FISH and FFPE tissue in the clinical diagnosis of ES/PNET, DSRCT, and CCS. Methods: Sixteen ES/PNETs, six DSRCTs, and six CCSs were studied. Three poorly differentiated synovial sarcomas, three alveolar rhabdomyosarcomas, and three neuroblastomas served as negative controls. Interphase FISH analysis was performed on FFPE tissue sections with a commercially available EWSR1 (22q12) dual colour, breakapart rearrangement probe. Results: One fused signal and one split signal of orange and green, demonstrating rearrangement of the EWS gene, was detected in 14 of 16 ES/PNETs, all six DRSCTs, and five of six CCSs, but not in the negative controls. Conclusions: Interphase FISH using this newly developed probe is sensitive and specific for detecting the EWS gene on FFPE tissues and is of value in the routine clinical diagnosis of ES/PNET, DSRCT, and CCS.

Bizarre parosteal osteochondromatous proliferation with a t(1;17) translocation

Bizarre parosteal osteochondromatous proliferation (BPOP) is a benign lesion that tends to recur repeatedly. Histologically, BPOP contains three components (cartilage, bone, and spindle cells) in differing amounts. The histological findings of BPOP are similar to those of florid reactive periostitis (FRP) and subungual (Dupuytren’s) exostosis. Some authors have postulated that all of these conditions are reactive proliferative lesions representing different phases of reactive processes. Whether BPOP is a reactive proliferative lesion or a neoplastic lesion, however, remains controversial. Recently, a t(1;17)(q32;q21) translocation in BPOP was detected using chromosome banding and fluorescence in situ hybridization (FISH) analyses. Here, we describe a 39-year-old Japanese female with BPOP arising in the proximal phalanx of her third toe. A cytogenetic analysis revealed a t(1;17)(q42;q23) translocation. The breakpoints in this case are located close to those of previously reported cases. These results suggest that t(1;17) is a distinct translocation of BPOP and that BPOP is a neoplastic lesion, rather than a reactive proliferative process.

Low grade malignant peripheral nerve sheath tumour: varied cytological and histological patterns

Background: A small number of malignant peripheral nerve sheath tumours (MPNSTs) are low grade, and the nature of these low grade tumours has never been systematically assessed. Aims: To describe the clinicopathological, immunohistochemical, and ultrastructural features of low grade MPNST and to discuss the main differential diagnoses. Methods: Four cases of low grade MPNST were studied, including one coexistent with neurofibromatosis type 1. The tumours were analysed with respect to nuclear atypia, cellularity, nuclear enlargement, hyperchromasia, mitotic rate, and necrosis. Immunohistochemistry was performed by standard techniques, and an ultrastructural study was performed on one tumour. Results: The ages of the patients ranged from 32 to 72 years (mean, 58). Two were male and two were female. Three tumours occurred in the deep tissue, including one in the retroperitoneum, and one was located in the dermal and subcutaneous tissue. The maximum diameters of the tumours ranged from 3.5 to 8.0 cm. Microscopically, all tumours showed moderate hypercellularity, an increased nuclear to cytoplasmic ratio, and hyperchromasia, but exhibited varied growth patterns, including those that were atypical neurofibroma-like, low grade fibromyxoid sarcoma-like, low grade epithelioid, and haemangiopericytoma-like. All tumours showed immunoreactivity for S-100 protein and vimentin. Conclusions: These findings suggest that careful clinical and histological evaluation, along with S-100 protein immunostaining, are essential for the accurate diagnosis of low grade MPNST.

Functional genome screen for therapeutic targets of osteosarcoma

Osteosarcoma (OS) is the most frequent primary malignant bone tumor of children and young adults. Although the introduction of combined neoadjuvant chemotherapy has markedly improved survival, the outcome of OS patients with distant metastasis and/or poor response to chemotherapy is still unsatisfactory. Therefore there is a need to develop new therapeutic agents that suppress OS cell proliferation with higher efficacy. The protein kinases are a family of genes that play critical roles in various signaling pathways. Some cancer cells show addiction to constitutive activation of certain signaling pathways for proliferation and survival. To identify new drug targets for OS, we screened a panel of small interfering RNAs (siRNAs) that target 691 genes encoding human protein kinases and related proteins. We found that different constructs of siRNA specifically targeting polo‐like 1 kinase (PLK1) significantly caused mitotic cell cycle arrest and subsequent apoptotic cell death in a variety of OS cell lines. siRNA targeting PLK1 also suppressed the growth of OS xenografts established in immunodeficient mice. Recently, phase I clinical trials of PLK1 chemical inhibitors have been reported. Our results indicate that PLK1 is a promising molecular target for pharmacologic intervention in OS. (Cancer Sci 2009; 100: 2268–2274)

Prognostic Implications of Glucose Transporter Protein-1 (Glut-1) Overexpression in Bone and Soft-Tissue Sarcomas

BACKGROUND The glucose transporter protein 1 (Glut-1) overexpression is associated with poor overall survival (OS) in various malignant tumors. The aim of this study was to investigate prognostic significance of Glut-1 overexpression in patients with bone and soft-tissue sarcomas. METHODS A total of 67 patients (mean age, 43 years; range, 8-79 years) with bone and soft tissue sarcomas were analyzed. Pathologic confirmation was observed from surgical specimens in all patients. Pathologic variables including tumor differentiation, necrosis, mitotic index, MIB-1 (Ki-67) grade and Glut-1 expression were assessed. Clinical characteristics and pathologic variables were determined by Kaplan-Meyer curve of OS after treatment. RESULTS Glut-1 overexpression was found in 56 patients (83%). The patients with Glut-1 overexpression showed significantly poor OS compared with those without Glut-1 overexpression (P = 0.029). The presence of metastasis, treatment without surgical resection, tumor differentiation, necrosis, mitotic index and MIB-1 grade were also significantly negative prognostic factors. The presence of metastasis was independently associated with poor OS (P = 0.031). CONCLUSIONS Assessment of Glut-1 expression prior to treatment has a predictive potential effect in patients with bone and soft-tissue sarcomas.